Inhibitors of farnesyl-protein transferase

Details Inhibitors of farnesyl-protein transferase Inhibitors of farnesyl-protein transferase

1996-09-12

2002-12-31

Seaman, D. Margaret (Department: 1625)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S419000, C546S147000, C548S492000

Reexamination Certificate

active

06500841

ABSTRACT:

BACKGROUND OF THE INVENTION
The Ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since Ras must be localized in the plasma membrane and must bind with GTP in order to transform cells (Gibbs, J. et al.,
Microbiol. Rev
. 53:171-286 (1989)). Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a “CAAX” or “Cys-Aaa
1
-Aaa
2
-Xaa” box (Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al.,
Nature
310:583-586 (1984)). Other proteins having this motif include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin.
Farnesylation of Ras by the isoprenoid farnesyl pyrophosphate (FPP) occurs in vivo on Cys to form a thioether linkage (Hancock et al.,
Cell
57:1167 (1989); Casey et al.,
Proc. Natl. Acad. Sci. USA
86:8323 (1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formation of a thioester on a Cys residue near a C-terminal Cys farnesyl acceptor (Gutierrez et al.,
EMBO J
. 8:1093-1098 (1989)); Hancock et al.,
Cell
57: 1167-1177 (1989)). Ki-Ras lacks the palmitate acceptor Cys. The last 3 amino acids at the Ras C-terminal end are removed proteolytically, and methyl esterification occurs at the new C-terminus (Hancock et al., ibid). Fungal mating factor and mammalian nuclear lamins undergo identical modification steps (Anderegg et al.,
J.Biol. Chem
. 263:18236 (1988); Farnsworth et al.,
J. Biol. Chem
. 264:20422 (1989)).
Inhibition of Ras farnesylation in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, N.J.) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al.,
Science
245:379 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids and the farnesyl pyrophosphate precursor. It has been shown that a farnesyl-protein transferase using farnesyl pyrophosphate as a precursor is responsible for Ras farnesylation. (Reiss et al.,
Cell
, 62: 81-88 (1990); Schaber et al.,
J. Biol. Chem
., 265:14701-14704 (1990); Schafer et al.,
Science
, 249: 1133-1139 (1990); Manne et al.,
Proc Natl. Acad. Sci USA
, 87: 7541-7545 (1990)).
Inhibition of farnesyl-protein transferase and, thereby, of farnesylation of the Ras protein, blocks the ability of Ras to transform normal cells to cancer cells. The compounds of the invention inhibit Ras farnesylation and, thereby, generate soluble Ras which, as indicated infra, can act as a dominant negative inhibitor of Ras function. While soluble Ras in cancer cells can become a dominant negative inhibitor, soluble Ras in normal cells would not be an inhibitor.
A cytosol-localized (no Cys-Aaa
1
-Aaa
2
-Xaa box membrane domain present) and activated (impaired GTPase activity, staying bound to GTP) form of Ras acts as a dominant negative Ras inhibitor of membrane-bound Ras function (Gibbs et al.,
Proc. Natl. Acad. Sci. USA
86:6630-6634(1989)). Cytosol localized forms of Ras with normal GTPase activity do not act as inhibitors. Gibbs et al., ibid, showed this effect in
Xenopus oocytes
and in mammalian cells.
Administration of compounds of the invention to block Ras farnesylation not only decreases the amount of Ras in the membrane but also generates a cytosolic pool of Ras. In tumor cells having activated Ras, the cytosolic pool acts as another antagonist of membrane-bound Ras function. In normal cells having normal Ras, the cytosolic pool of Ras does not act as an antagonist. In the absence of complete inhibition of farnesylation, other farnesylated proteins are able to continue with their functions.
Farnesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose. Reduction of farnesyl-protein transferase enzyme activity by adjusting the compound dose would be useful for avoiding possible undesirable side effects resulting from interference with other metabolic processes which utilize the enzyme.
These compounds and their analogs are inhibitors of farnesyl-protein transferase. Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of Ras, and other cellular proteins, with a farnesyl group. Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in vivo and inhibits Ras function. Inhibition of farnesyl-protein transferase is more specific and is attended by fewer side effects than is the case for a general inhibitor of isoprene biosynthesis.
Previously, it has been demonstrated that tetrapeptides containing cysteine as an amino terminal residue with the CAAX sequence inhibit Ras farnesylation (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al.,
PNAS
, 88:732-736 (1991)). It was, however, disclosed that tetrapeptides which further contained a cyclic amino acid residue, such as proline, had greatly reduced inhibitory activity when compared to tetrapeptides not containing a cyclic amino acid (Reiss et al., (1991). Tetrapeptide inhibitors may inhibit while serving as alternate substrates for the Ras farnesyl-transferase enzyme, or may be purely competitive inhibitors (U.S. Pat. No. 5,141,851, University of Texas).
It is, therefore, an object of this invention to develop non-peptide compounds which will inhibit farnesyl-protein transferase and the farnesylation of the oncogene protein Ras. It is a further object of this invention to develop chemotherapeutic compositions containing the compounds of this invention, and methods for producing the compounds of this invention.
SUMMARY OF THE INVENTION
The present invention includes partially reduced tetrapeptide analogs containing a cyclic amino acid which inhibit farnesyl-protein transferase (FPTase) and the farnesylation of the oncogene protein Ras, chemotherapeutic compositions containing the compounds of this invention, and methods for producing the compounds of this invention. It has been surprisingly found that these analogs containing a cyclic amino acid show FPTase inhibitory activity which is comparable to partially reduced tetrapeptide analogs which do not contain a cyclic amino acid. The invention also includes ester and lactone analogs which are prodrugs which deliver the active acid forms of the compounds to the intracellular compartment.
The compounds of this invention are illustrated by the formulae I and II:
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the inhibition of farnesyl-protein transferase and the farnesylation of the oncogene protein Ras. In a first embodiment of this invention, the inhibitors of farnesyl-protein transferase are illustrated by the formula I:
wherein:
R
1
and R
2
are independently selected from H, C
1-4
alkyl, C
1-4
aralkyl, —S(O)
m
—R
6
and
R
3
and R
4
are independently selected from: H; C
1-8
alkyl, alkenyl, alkynyl, or
 unsubstituted or substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with:
a) C
1-4
alkyl,
b) (CH
2
)
t
OR
6
,
c) (CH
2
)
t
NR
6
R
7
, or
d) halogen,
2) C
3-6
cycloalkyl,
3) OR
6
,
4) SR
6
, S(O)R
6
, SO
2
R
6
,
R
5
is hydrogen;
R
6
, R
7
and R
8
are independently selected from: H; C
1-4
alkyl, C
3-6
cycloalkyl, heterocycle, aryl, unsubstituted or substituted with:
a) C
1-4
alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
R
6
and R
7
may be joined in a ring, and
R
7
and R
8
may be joined in a ring;
R
9
is C
1-4
alkyl or aralkyl;
m is 0, 1 or 2;
t is 1 to 4;
X is O or H
2
;
Y is substituted or unsubstituted nitrogen containing C
4
-C
9
mono or bicyclic ring system wherein the non-nitrogen containing ring may be an aromatic ring, a C
5
-C
7
saturated ring or a heterocycle,

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